Adenovirus-mediated Downregulation Of The Ubiquitin Ligase RNF8 Sensitizes Bladder Cancer To Radiotherapy

Objective: Bladder cancer is the second most frequent cause of mortality among cancers of the genitourinary system and is the sixth most common malignancy in men worldwide.However,the dismal clinical context of bladder cancer necessitates the development of new therapeutic strategies.The clinical application of adenovirusmediated gene therapy displays the potential to substantially improve the therapeutic outcomes of patients,along with the possibility to preserve the bladder.The expression of the ubiquitin ligase RNF8,which can promote the DNA damage response(DDR),was increased in bladder cancer cells and that this change in RNF8 expression could be reversed by adenovirus-mediated sh RNA treatment.Therefore,the objective of this experiment is to explore the specific target therapeutic effects of RNF8 knockdown via adenovirus delivered sh RNA on the response to radiotherapy.Methods: Three bladder cancer cell lines,T24,BIU87 and 5637,were cultured and harvested in the logarithmic phrase of growth for Western blotting analysis in order to compare the amount of RNF8 expression,and it was followed by comparing the RNF8 expression of bladder tissues using this method.Then an immunofluorescence staining assay was used to detect endogenous RNF8 expression in bladder cancer cells.The presence of ionizing radiation-induced foci(IRIF)of RNF8 and ?-H2 AX,in response to DNA damage after exposure to 2 Gy of X-ray irradiation was evaluated.RNF8 expression was knocked down using adenovirus-mediated sh RNA in bladder cancer cell lines and then performed a colony formation assay.The efficient downregulation of RNF8 was confirmed by Western blotting.To determine the underlying mechanisms by which RNF8 knockdown conferred radiation sensitivity,immunocytochemistry was performed on the T24 bladder cancer cell line for a TUNEL assay.Sh RNF8-treated cells and their controls were either untreated or irradiated(5 Gy).Additionally,Acridine orange(AO)/propidium iodide(PI)double staining was performed to further investigate the apoptosis of T24 cells upon treatment with sh RNF8 and IR.To explore whether RNF8 participated in the DDR in bladder cancer cells,T24 cells were utilized to examine the process of post-irradiation DNA damage repair in the presence or absence of RNF8 knockdown by detecting ?-H2 AX foci at different time points.Cells infected with adenovirus-mediated sh RNF8 or sh Null vectors and control cells were treated with 5 Gy IR.To investigate the impact of RNF8 on H2 A and H2 B ubiquitination in bladder cancer cells,T24 cells transfected with sh RNF8 or sh Null were exposed to 5 Gy IR,and then cell lysates were prepared for Western blotting.To further investigate the role of RNF8 in DNA damage repair in bladder cancer cells,RNF8 were depleted using sh RNA,and immunofluorescence was performed to examine whether the formation of IRIF of various DNA damage signaling/repair proteins,including MDC1,53BP1,BRCA1 and RAP80,was RNF8-dependent.In vivo experiments were further performed to explore whether this effect of RNF8 could be observed in animal models.Tumors were established via subcutaneous injection of T24 cells into nude mice.When palpable tumors were established,the mice were randomized into sh RNF8-and sh Null-treated groups.Adenoviruses were injected into the tumors 3 times,on the 1 st,3 rd and 5 th days after tumor establishment.Western blots were performed to assess RNF8 expression in the tumor samples from the two groups on the 5 th day.Then,the mice were exposed to IR on the 6 th day,and tumor growth was monitored every three days.The therapeutic responses of the implanted tumors were assessed using histological staining with hematoxylin-eosin(H&E).Results: The expression of RNF8 in the three bladder cancer cell lines was upregulated compared to the control urothelial cell line SV-HUC-1(P < 0.05).RNF8 expression was also higher in tissue specimens from bladder cancer patients who underwent radical cystectomy than in normal control bladder cancer-adjacent tissues(P < 0.05).The immunofluorescent results showed that RNF8 clearly formed IRIF that co-localized with ?-H2 AX IRIF.The data from the colony formation assay showed that treatment with several doses of IR(2,4 and 8 Gy)did not appear to exert differential effects between untreated and sh Null-treated control cells at any IR dose;in contrast,adenosh RNF8-treated cells displayed a decreased survival rate compared to their controls.Altogether,these results suggested that the downregulation of RNF8 sensitized bladder cancer cells to radiotherapy.The non-irradiated bladder cancer cells displayed low levels of apoptosis.However,after exposure to 5Gy IR,T24 cells treated with sh RNF8 showed a significant increase in the proportion of TUNEL-positive cells compared to the control cells(P < 0.01).The results of AO/ PI double staining were consistent with those of the TUNEL assay;T24 cells treated with sh RNF8 showed a significant increase in the proportion of apoptosis cells compared to the control cells(P < 0.001).No difference in the number of ?-H2 AX IRIF was observed between the three groups at early time points(2 and 4 hours)post-IR.In contrast,at 24 hours post-IR,when most DSBs had been repaired in untreated and sh Null-treated control cells,the number of ?-H2 AX IRIF in sh RNF8 cells was significantly greater than that in control cells(P < 0.05).The levels of both Ub-H2 A and Ub-H2 B were increased after IR exposure in control cells;however,the Ub-H2A and Ub-H2B levels were decreased in RNF8-silenced cells compared with their mock sh Null-treated controls regardless of exposure to IR.In RNF8-silenced bladder cancer cells,?-H2 AX and MDC1,which function upstream of RNF8,clearly formed IRIF after radiotherapy.However,the downstream DNA repair proteins 53BP1,BRCA1 and RAP80 displayed abrogated recruitment and retention at DSB sites.RNF8 expression was decreased in the sh RNF8-treated group compared to the sh Null-treated group.Tumor growth in the sh RNF8-treated group was dramatically decreased compared to the sh Null-treated control group,though there was no significant difference in body size or weight between the two groups.The tumor tissue from the sh RNF8-treated group showed visible necrosis and apoptosis compared to the sh Null-treated control group.Conclusion: It was observed that the expression of the ubiquitin ligase RNF8 was increased in bladder cancer cells and that this change in RNF8 expression could be reversed by adenovirus-mediated sh RNA treatment.RNF8 promotes the DDR,and RNF8 participated in DNA damage repair after radiotherapy.This finding suggested that RNF8 might be a novel target for bladder cancer treatment.Moreover,it was found that RNF8 knockdown sensitized bladder cancer cells to radiotherapy,as demonstrated by reduced cell survival.Additionally,the absence of RNF8 induced a high rate of apoptosis and impaired double-strand break repair signaling after radiotherapy.This finding suggested that combination treatment of bladder cancer cells with sh RNF8 and IR can substantially improve the efficacy of radiotherapy by inducing apoptosis and that the DSB signaling and repair cascade was defective in the absence of RNF8.Also,it was confirmed that RNF8 participated in the radiotherapy-induced DNA damage response via the ubiquitination of H2 A and H2 B.Furthermore,experiments on nude mice showed that combining sh RNF8 treatment with radiotherapy suppressed implanted bladder tumor growth and enhanced apoptotic cell death in vivo.